All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Gene transfer vectors based upon the nonpathogenic parvovirus, adeno-associated virus (AAV), have recently emerged as promising tools for therapeutic gene transfer. Due to their relatively low immunogenicity and high transduction efficiency, AAV serotype 2 (AAV2) vectors have advanced to the forefront of human gene therapy. The recombinant AAV virus have been shown to transduce a wide array of cells and tissues in vitro and in vivo1-5. The AAV vectors are currently in use in Phase I/II clinical trials for gene therapy of cystic fibrosis, α-1 anti-trypsin deficiency, muscular dystrophy, factor IX-deficiency, and Parkinson's disease6, 7 8. However, some studies suggest that the transduction efficiency of AVV2 vectors in certain tissues types fall short of requirement for adequate level of gene expression9, 10.
The ubiquitin-proteasome pathway is reported to play an important role in the intracellular trafficking of AAV2 virus11, 12. It is reported that AAV2 capsids are phosphorylated at tyrosine residues by the epidermal growth factor receptor (EGFR) tyrosine kinase, but not at serine/threonine residues by casein kinase II (CKII) under cell-free conditions in vitro, and that AVV2 tyrosine-phosphorylation negatively affects viral intracellular trafficking and transgene expression in intact cells in vivo13. Recently, it has been reported that mutations of surface-exposed tyrosine residues on AAV2 capsids prevents ubiquitinalation of AAV2 thereby blocking proteasome-mediated degradation leading to generation of high-titer virus that is required for gene therapy14.
Thus, identification of amino acid motif within a protein sequence such as capsid protein of viruses would be extremely helpful in understanding viral trafficking and its stability in vitro and in vivo systems.